Effect of Human or Mouse IL-7 on the Homeostasis of Porcine T Lymphocytes

Abstract

Due to the inconsistent fluctuation of blood supply for transfusion, much attention has been paid to the development of artificial blood using other animals. Although mini-pigs are candidate animals, contamination of mini-pig T cells in artificial blood may cause a major safety concern. Therefore, it is important to analyze the cross-reactivity of IL-7, the major survival factor for T lymphocytes, between human, mouse, and mini-pig. Thus, we compared the protein sequences of IL-7 and found that porcine IL-7 was evolutionarily different from human IL-7. We also observed that when porcine T cells were cultured with either human or mouse IL-7, these cells did not increase the survival or proliferation compared to negative controls. These results suggest that porcine T cells do not recognize human or mouse IL-7 as their survival factor.

Keywords: Graft-versus-host disease; IL-7; Miniature swine; T-lymphocytes.

Figure 1. Phylogenetic tree analysis and structural comparison of human, porcine, and mouse IL-7. (A) The phylogenetic tree shows the relationship between human, porcine, and mouse IL-7 protein sequences. The evolutionary history was inferred using the neighbor-joining method. Evolutionary analyses were conducted in MEGA7. (B) The protein sequence homology among human, mouse, and mini-pig IL-7. The protein sequences of porcine, human, and mouse IL-7 were obtained in the FASTA format from the PubMed site. The sequences of the α-helix of human or mouse or porcine IL-7, which constitutes the binding site with IL-7 receptor, was examined by homologous analysis using Clustal Omega.

Figure 2. Effect of human or mouse IL-7 on the survival of porcine T cells. Porcine PBMCs were seeded in 48-well plates and treated with either human or mouse IL-7. On days 3, 7, and 14, cells were harvested, counted, and stained with anti-CD4 and anti-CD8 Abs and analyzed by flow cytometry. (A) The frequency of porcine CD4⁺ or CD8⁺ or CD4⁺CD8⁺T cells are shown by dot plots. (B-D) The numbers of CD4⁺ or CD8⁺ T cells were calculated based on flow cytometry data. We obtained similar results from other pigs of the same strain. Each experiment involved 7 independent experiments. Error bars represent SD. The statistical significance was measured by t-test.

Figure 3. Homeostatic proliferation of porcine T cells induced by either human or mouse IL-7 in vitro. The CFSE-labeled porcine PBMCs were seeded in 48-well plates and treated with either human or mouse IL-7. These cells were cultured and harvested on days 3, 7, and 14. The CFSE profiles were gated on CD4⁺ or CD8⁺ T cell staining. Histograms of CFSE-labeled CD4⁺ or CD8⁺ or CD4⁺CD8⁺T cells are shown. We obtained similar results from other pigs of the same strain. Each experiment involved 3 independent experiments.

Figure 4. Influence of human IL-7 on the survival of porcine T cells. Porcine PBMCs and mouse splenocytes were cultured in 48-well plates for 5 days. The frequencies and numbers of PI⁺ cells were measured by flow cytometry. (A) The fold change of percentage of PI⁺ lymphocytes are plotted at the indicated concentration of human IL-7. The percentage of PI⁺ porcine CD4⁺ (B) or CD8⁺ (C) or CD4⁺CD8⁺ (D) T cells is shown. Mouse splenocytes were used as a positive control in this experiment. These experiments were done in quadruplicates, in 3 independent experiments. Error bars represent SD. The statistical significance was measured by t-test.

^*p<0.05, ^**p<0.01, ^***p<0.005.

Figure 5. Influence of mouse IL-7 on the survival of porcine T cells. Porcine PBMCs and mouse splenocytes were cultured and treated with either media alone or different concentrations of mouse IL-7 for 5 days. The frequency and number of PI⁺ or PI⁻ cells were measured by flow cytometry. (A) The fold change of percentage of propidium iodide positive (PI⁺) lymphocytes are plotted at the indicated concentration of mouse IL-7. The percentage of PI⁺ porcine CD4⁺ (B) or CD8⁺ (C) or CD4⁺CD8⁺ (D) T cells is shown. Mouse splenocytes were used as a positive control in this experiment. These experiments were done in quadruplicates, in 3 independent experiments. Error bars represent SD. The statistical significance was measured by t-test.

^*p<0.05, ^**p<0.01.